This invention relates to the transformation of plant cells, and particularly to improved methods for the transformation of intact plant cells in tissue culture.
Much research in plant molecular biology is now directed to the improvement of plant varieties via use of recombinant DNA techniques. Historically, plant breeders used classical genetic techniques to identify, preserve and crossbreed varietal lines having desirable traits. More recently, new plant varieties were induced by chemicals or by radiation treatment to mutate plant cells which were then regenerated using tissue culture techniques. These random and unpredictable approaches have obvious drawbacks. By the use of recombinant DNA technology, specific genes producing specific proteins, such as those imparting insect resistance, may be introduced into a plant to produce a desired variety with a particular trait. One method for the introduction of recombinant DNA into plant cells is the microparticle bombardment method described by Sanford et al., Journal of Particle Science and Technology, 5:27-37, the disclosures of which are hereby incorporated herein by reference.
In the transformation of cells by microparticle bombardment introduction of recombinant DNA to impart a desired trait, it is important that the transformation be efficient, i.e., that large numbers of cells be transformed by the method, at least transiently, so that the function and effectiveness of the structural gene of interest and/or the regulatory sequences associated with the structural gene of interest can be evaluated by conventional analytical methods. In the past, efficiency of transformation of some cell lines, such as soybean tissue culture cells, has been undesirably low, so that gene products were not formed in sufficient amounts to be analyzed quantitatively by simple methods. Several different treatments were tried to increase the level of transient .gene activity. These have included treatment with various plant hormones, use of different cultivars of soybeans, and use of different transformation vectors. Disadvantages with each of these approaches continued to be low transient gene activity and a high degree of variability from experiment to experiment.